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Developmental toxicity / teratogenicity

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developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
23 January, 1992 - 9 March, 1993
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study was carried out under GLP and followed the appropriate guideline. The bodyweights were not measured with the frequency recommended in the guideline, however this is not deemed to affect the scientific integrity of the study.

Data source

Referenceopen allclose all

Reference Type:
study report
Report Date:
Reference Type:
study report
Report Date:

Materials and methods

Test guideline
according to
EPA OPP 83-3 (Prenatal Developmental Toxicity Study)
GLP compliance:
Limit test:

Test material

Test material form:
solid: particulate/powder
migrated information: powder
Details on test material:
- Name of test material (as cited in study report): Busan 11-M1
- Substance type: No data
- Physical state: White powder
- Analytical purity: 94.3
- Impurities (identity and concentrations): No data
- Composition of test material, percentage of components: No data
- Isomers composition: No data
- Purity test date: No data
- Lot/batch No.: 1-9769
- Expiration date of the lot/batch: No data
- Stability under test conditions: No data
- Storage condition of test material: No data

Test animals

New Zealand White
Details on test animals and environmental conditions:
- Source: Hazleton Research Products, Inc., Denver, Pennsylvania
- Age at study initiation: approximately 5 months
- Weight at study initiation: 2.344 - 3.710 kg
- Fasting period before study:
- Housing: Individually, stainless steel wire bottom cages suspended above a cage-board
- Diet (e.g. ad libitum): Purina Certified Rabbit Chow #5322 ad libitum
- Water (e.g. ad libitum): Municipal water ad libitum
- Acclimation period: approximately 6 weeks

- Temperature (°C): 20 - 22.2
- Humidity (%): 28-60%
- Air changes (per hr): 10 fresh air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hours light/dark cycle


Administration / exposure

Route of administration:
oral: gavage
other: 0.5% aqueous methylcellulose
Details on exposure:
An appropriate amount of the test material was weighed for each group and transferred to a mortar. The test material was triturated with a small amount of the vehicle until a slurry was formed. This was then transferred to a graduated cylinder and a sufficient amount of vehicle was added to attain an appropriate volume for mixing. The suspension was mixed on a mixer for approximately 5 minutes to reduce any large particles.
A magnetic stir bar was added and the mixture was stirred continuously throughout the sampling and dosing procedures. Dosing preparations were dispensed on a daily basis for dose administration. Dosing preparations were prepared twice during the study period and were stored at room temperature.

- Justification for use and choice of vehicle (if other than water): No data
- Concentration in vehicle: Each litre of vehicle was prepared by heating 1000 ml of deionised water to approximately 70 °C and gradually adding 5.0 g of the control material powder. The mixture was stirred until clear. The vehicle was prepared approximately once each week and stored refrigerated between periods of use.
- Amount of vehicle (if gavage): Dosage volume 1 ml/kg
- Lot/batch no. (if required): 50H0209
- Purity: 0.5% aquous methyl cellulose
Analytical verification of doses or concentrations:
Details on analytical verification of doses or concentrations:
Prior to initiation of dosing, duplicate aliquots taken from the top, middle and bottom of the preparations were tested for homogeneity. Two duplicate sets of aliquots were collected for the middle of the preparations for concentration and 14-day stability analysis. Duplicate sets of sample aliquots were collected form the middle of the dosing solutions at dosing initiation for concentration analysis. Analysis was carried out at EPL BioAnalytical Services, Inc.
The dosing preparations were homogenous, contained the amounts of test material specified by the protocol and were stable for 14 days.
Details on mating procedure:
- Impregnation procedure: artificial insemination
- A 0.25 – 0.5 ml aliquot of each diluted semen sample (collected from 10 resident males of the same strain and obtained from the same supplier as the females) was deposited into the anterior vagina of each female with a glass insemination pipette. Immediately following insemination each doe was administered an intravenous injection of human chorionic gonadotropin to ensure ovulation. The day of insemination was designated gestation day 0.
Duration of treatment / exposure:
From gestation days 7-19
Frequency of treatment:
Duration of test:
Dam gross necropsy examination performed on gestation Day 29
No. of animals per sex per dose:
20 females/dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: based on the results of a preliminary range-finding study
Five groups of 7 pregnant females were dosed with 20, 55, 90, 125, 160 mg/kg/day Busan 11-M1 daily from gestation day 7-19 by oral gavage. The animals were then observed until gestation day 29 at which point they were sacrificed and gross necropsy examination was performed. Maternal toxicity was exhibited at dose levels of 20 mg/kg/day and greater by mortalities and changes in the general clinical condition of the animals. No developmental toxicity was expressed at any dose level available for evaluation 20, 55, 90 mg/kg/day. Based on these results, the dose levels 2, 10, and 20 mg/kg/day were selected for the definitive developmental toxicity study.

- Rationale for animal assignment (if not random): computer randomisation procedure


Maternal examinations:
- Time schedule: twice daily
- Cage side observations included: mortality and moribundity

- Time schedule: On the first day of dosing, animals were observed one hour following dosing. On the second day of dosing, animals were observed one, two and four hours following dosing. For the remainder of the test, animals were observed one hour following dosing.

- Time schedule for examinations: recorded individually on gestation days 0, 7-20, 24 and 29

- Food consumption for each animal determined and mean daily diet consumption calculated as g food/animal/day and g food/kg body weight/day: Yes

- Sacrifice on gestation day 29
- Post mortem findings were correlated with ante mortem clinical finding as and any abnormalities were recorded.

Gross necropsy was performed on females which aborted or died during the course of the study. Maternal tissues were retained for possible future histopathological examination. The number and location of implantation sites and corpora lutea were recorded. Foetal finding for females which aborted were not included in any tabulation or statistical analysis.
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
Gravid uterine weight, net body weight (excluding the weight of the uterus and contents) and net body weight change were presented for each gravid female.
- Number of corpora lutea: Yes, in each ovary
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Other: Uteri with no macroscopic evidence of nidation were opened and subsequently placed in 10% ammonium sulfide solution for detection of early implantation loss.
Fetal examinations:
- External examinations: Yes: all per litter, including but not limited to eyes, palate and external orifices. Crown-rump measurements for late resorptions. Sex was determined.
- Soft tissue examinations: Yes: all per litter, heart and major vessels, kidneys
- Skeletal examinations: Yes: all per litter
- Head examinations: Yes: all per litter, brain was examined by a mid-coronal slice
All statistical tests were performed by a Digital MicroVAX 3400 computer with appropriate programming. All analyses were conducted using two-tailed tests for a minimum significance level of 5% comparing each treated group to the vehicle control group. Each mean was presented with the standard deviation and the number of animals used.
Foetal sex ratios: Chi-square test with Yate’s correction factor
Malformations and variations: Fishers Exact test
Early and late resorptions, dead foetuses, postimplantation losses: Mann-Whitney U-test
Corpora Lutea, total implantations, viable foetuses, foetal bodyweights, maternal bodyweights and weight changes, maternal net bodyweight changes and gravid uterine weights, maternal food consumption: ANOVA with Dennett’s test
Litter proportions of intrauterine data: Kruskal-Wallis test

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:
Maternal toxic effects:yes

Details on maternal toxic effects:
One animal in the 20 mg/kg/day group died on gestation day 16 and was internally normal. No clinical signs were noted in this female during the study. The death was consistent with the mortality observed in the range-finding study.
No other treatment related effects were found in any of the other animals.

Effect levels (maternal animals)

Key result
Dose descriptor:
Effect level:
10 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:

Results (fetuses)

Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
No efffects

Effect levels (fetuses)

Key result
Dose descriptor:
Effect level:
20 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: No effects observed at the highest dose level

Fetal abnormalities

Key result
no effects observed

Overall developmental toxicity

Developmental effects observed:
not specified

Any other information on results incl. tables

No further data

Applicant's summary and conclusion

No evidence of teratogenicity or developmental toxicity was seen in this study in the rabbit at dose levels sufficient to cause maternal toxicity.
Executive summary:

In a developmental toxicity study performed by Lamb (1993) groups of 20 pregnant female rabbits were dosed with 2, 10, 20 mg/kg bw/d Busan 11-M1 daily from gestation day 7-19 by oral gavage. The animals were then observed until gestation day 29 at which point they were sacrificed and gross necropsy examination was performed. One animal in the highest dose group died. No other effects attributable to treatment were observed on any of the other animals and no embryotoxic/teratogenic effects attributable to treatment were encountered. Based on the results of this study, the NOAEL for maternal toxicity was considered to be 10 mg/kg/day and the NOAEL for developmental toxicity was considered to be 20 mg/kg/day